NONO enhances mRNA processing of super‐enhancer‐associated GATA2 and HAND2 genes in neuroblastoma

Abstract

High‐risk neuroblastoma patients have poor survival rates and require better therapeutic options. High expression of a multifunctional DNA and RNA‐binding protein, NONO, in neuroblastoma is associated with poor patient outcome; however, there is little understanding of the mechanism of NONO‐dependent oncogenic gene regulatory activity in neuroblastoma. Here, we used cell imaging, biochemical and genome‐wide molecular analysis to reveal complex NONO‐dependent regulation of gene expression. NONO forms RNA‐ and DNA‐tethered condensates throughout the nucleus and undergoes phase separation in vitro , modulated by nucleic acid binding. CLIP analyses show that NONO mainly binds to the 5′ end of pre‐mRNAs and modulates pre‐mRNA processing, dependent on its RNA‐binding activity. NONO regulates super‐enhancer‐associated genes, including HAND2 and GATA2. Abrogating NONO RNA binding, or phase separation activity, results in decreased expression of HAND2 and GATA2. Thus, future development of agents that target RNA‐binding activity of NONO may have therapeutic potential in this cancer context. image NONO protein correlates with poor outcome in neuroblastoma. NONO binds 5′ ends of introns in oncogenic genes, including super‐enhancer‐regulated GATA2 and HAND2, enabling proper RNA processing, via RNA binding and phase separation activities. In neuroblastoma, the multifunctional protein NONO binds to the 5′ intronic regions of pre‐mRNA in non‐paraspeckle nuclear puncta. Some NONO puncta correspond to transcription sites for super‐enhancer regulated GATA2 and HAND2 genes, where NONO enhances RNA processing at the 5′ end, leading to robust gene expression. NONO RNA binding and low‐complexity domains are required for its activity that is also diminished by BRD4 inhibitors, suggesting the puncta are phase separated condensates.